The glyceraldehyde-3-phosphate dehydrogenase promoter, P(GAP), was employed to direct the constitutive expression of recombinant human serum albumin (HSA) in Hansenula polymorpha. A set of integration vectors containing the HSA cDNA under the control of P(GAP) was constructed and the elemental parameters affecting the expression of HSA from P(GAP) were analyzed. The presence of a 5'-untranslated region derived from the HSA cDNA and the integration of the expression vector into the GAP locus were shown to improve the expression of HSA under P(GAP). Glycerol supported a higher level of HSA expression from P(GAP) along with a higher cell density than either glucose or methanol. The growth at high glycerol concentrations up to 12% did not cause any significant repression of the cell growth. A high cell density culture, up to 83 g l(-1) dry cell weight with a HSA production of 550 mg l(-1), was obtained in less than 32 h of cultivation in a fed-batch fermentation employing intermittent feeding with 12% glycerol. The GAP promoter-based HSA expression system showed a higher specific production rate and required a much simpler fermentation process than the MOX promoter-based system, demonstrating that P(GAP) can be a practical alternative of the MOX promoter in the large-scale production of HSA from H. polymorpha.

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Proteomic responses of methylotrophic yeasts (Hansenula polymorpha DL1 and A16) to growth medium tuning by carbon source shift (glycerol-->methanol) were monitored and analyzed by two-dimensional gel electrophoresis. Through comparative analyses of two-dimensional gels, intracellular yeast proteins with complex expression patterns were systematically sorted into: (1) proteins that are commonly expressed with comparable high abundance in both strains; (2) strain-specific proteins that are expressed at high level only in a particular strain; (3) strain-specific and methanol-induced proteins that are expressed only in the presence of methanol; and (4) strain-specific and constitutively-expressed proteins that are expressed consistently irrespective of carbon source shift without extreme change in expression level. Among the DL1-specific proteins belonging to group four, the four proteins showing the highest expression levels in the course of the fermentation process were identified as: glucose-6-phosphate dehydrogenase, isocitrate lyase, succinyl-CoA synthetase, and glycerol-3-phosphate dehydrogenase. From these results, it is suggested that DL1 has distinct metabolic characteristics including enhanced metabolic activities both in glycerol uptake and the glyoxylate bypass cycle, as compared to A16. This is likely to explain why the DL1 strain shows a significantly higher rate of glycerol and methanol consumption during the fermentation process. Our systematic approach to the analysis of proteomic responses and the detailed analysis results reported here will be useful to better understand the global physiology of H. polymorpha, as proteome databases for various methylotrophic yeasts are established.

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Aims: To screen and clone a novel enzyme with specific activity for the resolution of (R)-b-acetylmercaptoisobutyrate (RAM) from (R,S)-b-acetylmercaptoisobutyrate [(R,S)-ester]. Methods and Results: A micro-organism that produces a novel esterase was isolated and identified as the bacterium Burkholderia cepacia by using the analysis of cellular fatty acids, Biolog automated microbial identification/characterization system, and 16S rRNA gene sequence analysis. A novel esterase gene was cloned from the chromosomal DNA of B. cepacia and was designated as cpoA. The cpoA encodes a polypeptide of 273 amino acids which shows a strong sequence homology with many bacterial nonhaeme chloroperoxidases. In addition, a typical serine-hydrolase motif, Gly-X-Ser-X-Gly, and the highly conserved catalytic triad, Ser95, Asp224, and His253, were identified in the deduced amino acid sequence of cpoA by multiple sequence alignment. Conclusion: The cpoA cloned from B. cepacia encodes a novel esterase which is highly related to the nonhaeme chloroperoxidases. Significance and Impact of the Study: This is the first report that describes the isolation and cloning of a serine esterase gene from B. cepacia, which is useful in the chiral resolution of (R,S)-ester. The cloned gene will allow additional research on the bifunctionality of the enzyme with esterase and chloroperoxidase activity at the structural and functional levels.